The long-term objective of this project is to understand how the process of differentiation prevents over-proliferation during embryogenesis. More specifically, the transcription factor and onco-protein Myc is required for proliferation during embryogenesis, and yet an over-abundance of Myc protein induces tumors;how do embryonic cells achieve the correct balance of Myc function? Myc is a transcriptional regulator with the ability to activate and repress transcription of different targets. Transcriptional activation by Myc requires its binding to DNA, and though repression by Myc is not necessarily mediated by its binding to DNA, a Myc mutant that is unable to bind DNA is unable to repress. One of the first identified targets of Myc repression was the c-myc gene itself, and all human tumor-derived cell lines tested are defective for this auto-repression of myc. In an effort to understand the mechanism of myc auto-repression, along with Eric Wieschaus and Michael Cole at Princeton University, I developed a novel genetic screen using Drosophila melanogaster. We found that mutations in Polycomb, a chromatin binding repressor required for maintenance of cell fates, eliminate auto-repression of the Drosophila myc gene, and also the majority of repression mediated by ectopic Myc in the embryo. Mutations in Polycomb also led to increased expression of many of Myc's targets of activation, suggesting a link between Polycomb, repression by Myc and transactivation by Myc. Though biologically important, the link between Polycomb and Myc is not known to be direct or indirect. In fact, in the Drosophila embryo, Myc induces the expression of gene products functionally related to Polycomb: Su(z)2, Esc and Pho (homologs of mammalian bmi-1, eed and YY1, respectively). In this proposal, I hypothesize that Myc activation of Polycomb Group gene products Su(z)2, Esc and Pho causes Myc's downstream repression of targets, and that the relative abundance of these repressors throughout embryogenesis affects Myc trans-repression of targets. To test this hypothesis, my specific aims are: (1) to determine whether Myc binds directly to Su(z)2, pho and/or esc genes, causing their activation;(2) to determine whether Su(z)2 and/or Esc are required for repression by Myc;and (3) to determine what proteins associate with Myc throughout embryogenesis. Genetic alterations that lead to increased Myc expression cause uncontrolled proliferation and tumorigenesis. In an embryo, however, Myc levels are high and proliferation is controlled. The research described in this proposal will provide insight into the mechanisms operating in embryonic cells that maintain normal proliferation and prevent tumorigenesis. The genetic and molecular biological tools available in Drosophila will rapidly provide a depth of understanding that is difficult, if not impossible, to obtain using mammalian systems. PUBLIC HEALTH RELEVANCE: Organisms require cellular proliferation for normal processes such as growth during development and blood production. A protein called Myc is required for normal proliferation, although Myc causes cancer when over-abundant. This proposal investigates mechanisms of Myc activity in a tissue with naturally high levels of Myc.